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Oncotarget, December, Vol.2, No 12 



Antitumoral activity of allosteric inhibitors of protein kinase 
CK2 

Virginie Moucadel 1 ' 23 *, Renaud Prudent 1 ' 23 *, Celine F. Saute! 123 , Florence 
Teillet 123 , Caroline Barette 4 , Laurence Lafanechere 4 , Veronique Receveur- 
Brechot 5 and Claude Cochet 1 ' 23 

1 From INSERM, U1036, Biology of Cancer and Infection, Grenoble, F-38054, France 

2 CEA, DSV/iRTSV, Biology of Cancer and Infection, Grenoble, F-38054, France 

3 UJF-Grenoble 1, Biology of Cancer and Infection, Grenoble, F-38041, France 

4 CEA, iRTSV/CMBA, Grenoble , F-38054, France 

5 IMR Laboratory CNRS UPR3243, IMM, F-13402 Marseille cedex 20, France 

Both authors contributed equally to this work 
Correspondence to: Claude Cochet, email: claude.cochet@cea.fr 
Keywords: Protein-kinase CK2, Inhibitors, Azonaphthalene, Cancer, SAXS 

Received: November 24, 201 1 , Accepted: November 29, 201 1, Published: December 14, 201 1 

Copyright: © Moucadel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which 
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 

ABSTRACT: 

Introduction : Due to its physiological role into promoting cell survival and its 
dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological 
target for development of chemical inhibitors. We report the discovery of 
azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First, 
we demonstrated that CK2 inhibition (IC 50 = 0.4 pM) was highly specific, reversible 
and non ATP-competitive. Small Angle X-ray Scattering experiments showed that 
this inhibition was due to large conformational change of CK2a upon binding of 
these inhibitors. We showed that several compounds of the family were cell-potent 
CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally, 
in vitro and in vivo assays showed that these compounds could decrease U373 cell 
tumor mass by 83 % emphasizing their efficacy against these apoptosis-resistant 
tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no 
effect in colony formation and tumor regression assays. These findings illustrate the 
emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the 
development of novel allosteric CK2 inhibitors. 

Background : CK2 is an emerging therapeutic target and ATP-competitive 
inhibitors have been identified. CK2 is endowed with specific structural features 
providing alternative strategies for inhibition. 

Results : Azonaphthalene compounds are allosteric CK2 inhibitors showing 
antitumor activity. 

Conclusion : CK2 may be targeted allosterically. 

Significance : These inhibitors provide a foundation for a new paradigm for 
specific CK2 inhibition. 



INTRODUCTION 

Protein kinase CK2 plays critical roles in cell 
growth and differentiation, apoptosis and oncogenic 
transformation [1, 2]. Aberrant CK2 kinase expression 
was associated with unfavorable prognostic markers in 



prostate cancer [3] and in acute myeloid leukemia [4] 
implicating CK2 in tumor formation and recurrence. Its 
dysregulation in many other cancers together with its dual 
function in promoting cell growth and in suppression of 
apoptosis may be particularly relevant to its oncogenic 
potential [5]. Further relevance for its role in cancer is 



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indicated by its mediation of resistance to chemotherapy- 
induced apoptosis [6]. Involvement of CK2 in other 
diseases such as glomerulonephritis [7] or viral infections 
(HIV, CMV, HPV, PV...) has been reported [8]. Based 
on these observations, CK2 is now considered to be a 
relevant physiopathological target amenable to therapeutic 
intervention therefore supporting the identification and the 
characterization of chemical inhibitors [9, 10]. Extensive 
efforts have been focused on developing CK2 inhibitors 
and several active, and in certain cases selective, ATP 
site-directed compounds have been identified [11]. These 
include condensed polyphenolic compounds such as 
emodin and derivatives of hydroxycoumarins (3-carboxy- 
4(lH)-quinolone),the indoloquinazoline derivative (IQA), 
tetrabromocinnamic acid (TBCA), 4,5,6, 7-tetrabromo-l- 
benzotriazole (TBB) and pyrazolo[l,5-a][l,3,5]triazine 
derivatives [12]. Recently, our group and others reported 
the identification of very efficient CK2 inhibitors with 
good in vivo potency [13-15]. 

Beside ATP-competitive inhibitors binding to 
the canonical ATP-site, small molecules targeting 
different surfaces of kinases [16-18], including CK2 
[19, 20] have been identified. Some of them bind to the 
hydrophobic CK2B-binding cavity on CK2a, possibly 



inducing an inactive conformation [21]. Indeed, an 
inactive conformation of the catalytic CK2a subunit 
was recently reported [22]. In this CK2a structure, it has 
been suggested that the binding of small molecules to the 
CK2B-docking site have an inhibitory impact on CK2a by 
promoting its inactive conformation [21, 22]. Altogether, 
these observations suggest the existence on CK2 of 
different exosites distinct from the catalytic cavity that 
can be targeted by small molecules to achieve functional 
effects [19]. 

Using an automated screening, we have identified 
azonaphthalene derivative compounds as new highly 
potent CK2 inhibitors. 

We report that azonaphthalene derivatives are 
specific non ATP-competitive CK2 inhibitors. Small 
Angle X-Ray Scattering analysis showed a major 
conformational change of the kinase upon inhibitor 
binding, Furthermore, several compounds of the family 
are cell-permeable CK2 inhibitors promoting cell cycle 
arrest of human glioblastoma U373 apoptosis-resistant 
cells. Finally, we demonstrate that these compounds 
decrease tumorigenesis in vitro and exhibit in vivo efficacy 
in tumor growth assays. 

These results show that a relevant allosteric 




Recovery 



MHmM) 




1/[ATP] ((jM- 1 ) 




1/[Peptide] (mM' 1 ) 



Figure 1: Characterization of compound 1 as a reversible non-competitive CK2 inhibitor. A. Inhibition of the catalytic 
activity of recombinant 36ng CK2a (♦) or 60ng CK2 holoenzyme (a 22 ) (■) by increasing concentrations of compound 1. B. Reversibility 
of compound 1 inhibition. CK2a (2|ig) was incubated with 25uM compound 1 following by a gel-filtration chromatography. CK2 kinase 
activity in input and flow-through were then assayed. C and D. Lineweaver-Burk inhibition plots of human recombinant CK2a by 
compound 1. CK2 kinase activity was determined as described in the experimental section in the absence (♦) or in the presence of 2.5 (■), 5 
( A ) and 7.5 (X) uM compound 1 with various concentration of ATP (C) or peptide substrate (D). The data represent means of experiments 
run in triplicate with SEM never exceeding 10%. 



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inhibition of CK2 activity can be achieved with non-ATP 
competitive inhibitors expanding the options to modulate 
this enzyme. 

RESULTS 



Identification of a new potent CK2 inhibitor 
scaffold 

The 2,860 compounds from the National Cancer 
Institute Developmental Therapeutics Program small 
molecule library were screened in an automated 
luminescence-based in vitro kinase assay against the 
human recombinant CK2 catalytic subunit CK2a as 
previously published [21]. As a primary screen, CK2 
kinase inhibitory activity was determined by measuring 
the percentage of inhibition at a compound concentration 
of 15 uM, using TBB and DMSO as positive and negative 



controls respectively. A secondary screen performed at a 
compound concentration of 1.5 |iM allowed the isolation 
of 1 1 hits. Hit validation was performed at concentrations 
of 1.5 uM using standard radiometric kinase assay 
with high ATP concentrations (100 uM, K m for ATP of 
recombinant CK2: 25 (0.M) to enhance the probability of 
isolating non-ATP competitive compounds. This led to 
the characterization of compound 1 as micromolar CK2 
inhibitor (Figure SI). Compound 1 displays an IC 50 ~ 5 
(0.M on CK2a and is equally potent on CK2a alone or 
complexed with its regulatory CK2[3 subunit (Figure 1A). 

Given that this active compound is hydrophobic, 
planar and rigid with peripherical polar groups, we 
investigated whether it could behave as promiscuous 
inhibitors [23], acting via an aggregation mechanism [24, 
25]. Since aggregate-forming inhibitors often display 
steep dose-response curves and high Hill coefficients 
[26, 27], we performed dose-response curves for CK2 
inhibition. It was observed that compound 1 displayed 
standard dose response curves with Hill slopes ranging 



o 
n 

> 

« 1 
re 

3 

■a 

tf> 0.5 
<D 

0£ 



51 



101 151 201 

Mutated residue 



251 



301 



3 

■2, 

-=io- : 



10" 



10- ; 





0.00 



0.10 



0.20 0.30 
q (A" 1 ) 



0.40 



CK2a 



CK2a«1 



Figure 2: Structure of CK2a and CK2a-l complex as determined using SAXS Ab initio shape restoration. A. Inhibitory 
effect of compound 1 on WT and a panel of mutants with the following residues mutated to alanine (10, 24, 61, 71, 74, 76, 80, 123, 138, 
160, 178, 195, 253, 268, 308, 311). Residual activity ratio is defined as the normalized activity of mutant CK2a divided by the normalized 
activity of wild-type CK2 in presence of 5|xM compound 1. B. Experimental scattering spectrum of CK2a (black line, above) and of CK2a- 
1 complex (black line, below), and theorical spectra of CK2a calculated by CRYSOL using the crystal structure of CK2a (PBD ID 1PJK) 
as template (red line), and of the shape calculated by GASBOR for CK2a (blue line) and CK2a-l complex (green line). The curves have 
been artificially shifted for better lisibility. C. Typical calculated shape obtained by GASBOR (green envelope) of CK2a (left panel) and 
CK2a-l complex (right panel), superimposed with the atomic structures of CK2a (PDB ID 1PJK). The atomic structure is in red ribbon 
representation. 



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between 1.1 to 1.3 indicating that the compound likely 
binds its target enzyme through a classical 1 : 1 inhibition 
mechanism. Moreover size-exclusion chromatography 
experiments indicated that CK2a inhibition by compound 
1 is reversible (Figure IB), a feature that is rarely 
associated with aggregating compounds. Taken together, 
this rules out compound 1 as a promiscuous aggregator. To 
define the mode of CK2 inhibition by compound 1, we 
performed steady-state kinetic analysis. Lineweaver-Burk 
inhibition plots showed that inhibition pattern of CK2a 
by compound 1 was non competitive toward both ATP 
and peptide substrate (Figure 1C and ID). Of note, at high 
peptide substrate concentrations the plots were non-linear 
suggesting that in the presence of saturating substrate 
concentrations, the enzyme may be more susceptible to 

A 



DMSO TBB 1 1 

48h 24h 24h 48h 




inhibition by compound 1 . 

SAXS analysis of CK2a-l complex 

To determine the binding site of compound 1, we 
tested its effect on a series of CK2a mutants (Figure 
2A). This revealed that CK2a mutated on K78A, R80A 
or L178A were more resistant to compound 1 inhibition, 
suggesting that these residues located on helix aC and 
on the activation segment are part of the compound 1 
binding site. To get better insights into the molecular 
interactions between CK2a and compound 1, we tried 
to obtain X-ray crystallographic co-structure of CK2a- 
1 complex. However, despite several crystallization 

B 

DMSO TBB 1 23 



pSerl3-Cdc37 

Cdc37 

Tubulin 





0 10 20 30 40 50 60 70 80 90 100 
Compound Concentration (u.M) 



100 



■40 



I 20 




DMSO 
0.5% 



□ pGL5-GFP 

□ pGL5-CK2a 



TBB 

50 uM 



1 

50 \lM 



120 
100 
80 
60 
40 



□ DMSO (0.5%) Dl^OuM) 
0 TBB (50 □ 23(50^) 



1 1*1 



A549 H1299 LnCap MDA231 MCF-7 
Cell Lines 



Figure 3: Compound 1 is a cell-potent CK2 inhibitor and decreases cell viability in a CK2 dependent manner. A. HeLa 
cells were plated and transfected with the CK2 activity reporter plasmid. One day after, medium was replaced with medium containing 
increasing amounts of compounds and incubated for 24h or 48h. Then, cells were collected and the reporter phosphorylation status was 
measured from whole cell extracts. Experiment was repeated 3 times. B. U373 cells were plated one day prior inhibitor addition. Twenty 
four hours after compound addition, cells were collected and phospho-Cdc37, Cdc37 and tubulin levels were measured by immunoblotting. 
Experiment was repeated twice. C. One day after plating, U373 cells were treated with increasing concentrations of TBB, 1, 23 or DMSO. 
Two days after, living cells were counted as described. Results are given relative to the luminescence recorded for DMSO. Experiment was 
done in triplicate and repeated twice. D. HeLa cells were transfected with a CK2rx or a GFP-expressing plasmid. One day later, medium 
was replaced with medium containing 0.5% DMSO or 50 uM TBB, 1. Two days after, living cells were counted as in C. Results are given 
relative to the luminescence recorded for DMSO in each condition. Experiment was done in triplicate and repeated twice. E. One day after 
plating, cells were treated with 50 uM TBB, 1, 23 or 0.5% DMSO. Two days after, living cells were counted as in A. Results are given 
relative to the luminescence recorded for DMSO in each cell lines. Experiment was done in triplicate and repeated twice. 



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screening campaigns, we were unable to get diffracting 
crystal containing CK2a complexed with compound 1 . 

Therefore, we performed Small Angle X-Ray 
Scattering (SAXS) experiments on rhCK2a AC (l-335) 
and rhCK2a AC (l-335)-l complex (Figure 2B). SAXS 
is indeed a very appropriate tool to assess the structural 
properties (dimensions, flexibility and overall shape) of 
proteins in solution. The Guinier law allows the scattered 
intensities to be approximated, at low scattering angles 
to determine the radius of gyration (R G ). When applied 
to CK2a, R values calculated for different protein 
concentrations revealed that upon compound 1 binding, 
R Q (extrapolated to zero concentration) increases from 
32.8±1.2 A to 35.6±1.1 A at 20°C and the corresponding 



Maximal diameter (Dmax) increases from68±3 Ato96±4 
A. As probed by kinase assay, compound 1 inhibition 
is reversible and Kratky plot of CK2a-l SAXS data 
exhibits a bell-shaped profile typical of globular protein. 
Thus, a denaturation of CK2a by compound 1 can be 
ruled out. The overall shape of rhCK2a AC (l-335) and 
rhCK2a AC (l-335)-l complex were calculated ab initio 
using the program GASBOR (Figure 2C). Different 
runs gave similar shapes. Averaged calculated shape of 
rhCK2a AC (l-335)-l complex superimposed with the 
X-ray structure of rhCK2a AC (l-335) (PDB ID 1PJK) 
shows that CK2a undergoes a conformational change, 
leading to a distorted shape. In this conformation, CK2a 
could be inactive due to non-optimal spatial arrangement 



Table 1: Features that confer inhibitory potency to compound 1. A. CK2 kinase assays were performed in the absence or 
presence of 10 uM compound. Results are expressed as a percentage of the control activity without inhibitor. B. Compounds that are cell 
permeable CK2 inhibitors. CK2 inhibition was monitored as in 





A 


B 




Compound 








4 | 






7 


8 


1 1 






4 


CK2 activity (%) 


1 


N=N 


OH 


H 


SO3H 


H 


N0 2 


H 


H 


N=N 


OH 


H 


H 


80 




N=N 


OH 


H 


SO3H 


H 


H 


H 


H 


N=N 


OH 


H 


H 


18.3 




N=N 


OH 


H 


SO3H 


H 


H 


H 


H 


N=N 


OH 


H 


H 


3.9 


1 4 


N=N 


OH 


H 


SO3H 


H 


N0 2 


H 


H 


OH 


N=N 


H 


H 


0.2 




N=N 


OH 


H 


SO3H 


H 


H 


H 


H 


OH 


N=N 


H 


H 


14.2 




N=N 


OH 


H 


SO3H 


N0 2 


H 


H 


H 


OH 


N=N 


H 


H 


1.3 


7 


NH 2 


OH 


H 


SO3H 


H 


H 


H 


H 


none 


83 


8 


N=N 


OH 


H 


H 


H 


H 


H 


H 


N=N 


H 


H 


H 


68.5 




N=N 


OH 


H 


SO3H 


H 


H 


H 


H 


A 


15.8 


10 


N=N 


OH 


H 


SO3H 


H 


H 


H 


H 


B 


48.7 


11 


N=N 


OH 


H 


SO3H 


H 


H 


H 


H 


OH 


N=N 


COOH 


H 


1.2 


12 


N=N 


OH 


S0 3 H 


H 


H 


SO3H 


H 


H 


N=N 


H 


H 


H 


28.5 


13 


N=N 


OH 


H 


H 


H 


SO3H 


H 


SO3H 


N=N 


H 


H 


H 


58.3 


14 


H 


N=N 


H 


H 


H 


SO3H 


H 


H 


N=N 


OH 


H 


H 


20.2 


15 


N=N 


H 


H 


H 


H 


H 


H 


SO3H 


N=N 


OH 


H 


H 


14.1 


16 


N=N 


H 


H 


H 


H 


H 


SO3H 


H 


N=N 


OH 


H 


H 


12 


17 


N=N 


H 


H 


H 


H 


SO3H 


H 


H 


N=N 


OH 


H 


H 


7.4 


18 


N=N 


H 


H 


H 


SO3H 


H 


H 


H 


N=N 


OH 


H 


H 


4.5 


19 


N=N 


OH 


H 


H 


H 


SO3H 


H 


H 


N=N 


H 


H 


H 


0.8 


20 


N=N 


OH 


SO3H 


H 


H 


SO3H 


H 


H 


N=N 


H 


H 


SO3H 


7.6 


21 


N=N 


OH 


H 


H 


H 


SO3H 


H 


SO3H 


N=N 


H 


H 


SO3H 


25.7 


22 


OH 


N=N 


H 


H 


SO3H 


H 


H 


H 


N=N 


H 


H 


SO3H 


20.9 


23 


N=N 


H 


H 


NH 2 


H 


SO3H 


H 


H 


C 


10 



A : p-cresol; B : 3-methyl-l-phenyl-lH-pyrazol-5-ol ; C : 4-phenylphenol 



B 



uM 


24h 


48 h 


50 


100 


200 


50 


100 


200 


1 


+ 


+ 


+ 


+ 


+ 


+ 














+ 


4 






+ 




+ 


+ 








+ 




+ 


+ 








+ 


ND 


ND 


ND 




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of its catalytic site. Alternatively, some domain movement 
may be impaired impeding catalysis. 

Effect of compound 1 on cellular CK2 kinase 
activity 

To evaluate the efficacy of compound 1 to target CK2 
into living cells, we used a cellular CK2 activity assay 
[28]. Compound 1 tested at increasing concentrations for 
24 or 48 h was active on cellular CK2 activity (Figure 
3A). This was also confirmed by immunoblotting 
using a phosphospecific antibody recognizing Cdc37 
phosphorylated on Serl3 which is specifically targeted 
by CK2 [29]. Thus, Serl3-Cdc37 phosphorylation 
status can be used as a surrogate cellular CK2 activity 
assay [29]. We found that under similar conditions (50 
|j,M, 48h incubation), compounds 1 like TBB, reduced 
drastically Cdc37 phosphorylation on Serl3. Compound 
23, an analogue of compound 1 which is known to be cell- 
permeable [30] was inactive both on recombinant CK2a 
and on cellular CK2 activity, (Figure 3B). 

Compound 1 decreases cell viability in a CK2- 
dependent manner 

CK2 inhibitors are known to decrease cell viability. 
Thus, we measured HeLa cell viability after 48h treatment 
with increasing amount of TBB or compound 1 . Compound 
23 was used as negative control. Viability of HeLa cells 
exposed to compound 1 or TBB showed similar decreased 
viability (IC 50 3 3 |iM and 36 (xM respectively) (Figure 
3C). To determine whether the reduction of cell viability 
triggered by compound 1 was mediated by CK2 inhibition, 
we transfected HeLa cells with a CK2a or a GFP- 
expressing plasmid. Cells expressing exogenous CK2a 
displayed a better survival when treated with compound 
1 than control cells (Figure 3D). Then, we measured the 
effect of compound 1 on the viability of different known 
tumor cell lines (Figure 3E). At a concentration of 50 uM, 
compound 1 decreased viability of most tested cell lines 
and was even more efficient than TBB, especially in p53 
mutant cell lines like U373 and to a lesser extent HI 299 
and MDA231. This decreased viability was correlated 
with a down regulation of CK2 activity attested by Cdc37 
Serl3 phosphorylation which was drastically reduced 
in U373 cells exposed to compound 1 (Figure 3B). In 
contrast, this reduction of Cdc37 Serl3 phosphorylation 
was not observed with 50 (.iM TBB or compound 23. 

Structure Activity Relationship of azonaphthalene 
compounds on CK2a 

To define the features that confer inhibitory potency 
to compound 1, we tested several derivatives in vitro and 



in the context of a complex cellular milieu (Table 1 and 
Table SI). First, compounds showing a CK2 inhibiting 
activity in vitro were tested at increasing concentrations 
for 24 or 48 h in the cellular CK2 assay. Five compounds 
inhibited CK2 in this assay: 1, 3, 4, 5 and 6 (Figure 3A 
and 3B, Table IB). The most potent compounds were: 1 
> 4, 5 >3 > 6. 

Given that this class of compound possess a 
symetrical scaffold made of two naphthyl parts (A and 
B) bound by an azo function, we first investigated the 
requirement of part A. Compound 6, which possesses a 
nitro function at position A5 was fully active both in vitro 
and in the CK2 cellular assay. Moreover, compound 2 
which lacks nitro function at position A6 remains active 
in vitro and compound 3 (which is zinc complex of 
compound 2) remains active in our cellular assay. Since 
nitro function is not necessary, it will be possible to remove 
it thereby avoiding possible carcinogenic metabolite. 
To gain insight into the requirements of the hydroxyl 
function in position A2 and the sulfonic acid in position 
A4, several derivatives of compound 1 were tested. 
Compound 8 which lacks the nitro, hydroxyl and sulfonic 
acid functions was notably inactive. This indicates a clear 
requirement for hydroxyl and/or sulfonic acid functions. 
Position variations of sulfonic acid function (compounds 
14-18) lead to in vitro active compounds, indicating a 
requirement for sulfonic acid function. However none 
of these compounds are active in the CK2 cellular assay. 
Thus, hydroxyl function at position A2 and sulfonic acid 
at position A4 is, up to now, the optimal combination to 
get cell-potent compounds. Moreover, both compounds 3 
and 5 are active both in vitro and in a CK2 cellular assay, 
indicating some tolerance in the position of the azo linker. 

We next investigated the requirements of part B. 
Removal of this moiety is detrimental (compound 7). This 
indicates that this part of the molecule possesses functions 
conferring activity both in vitro and in our CK2 cellular 
assay. Testing of compound 1 1 reveals that an additional 
carboxylic function enhances the in vitro activity. 
Compounds 9 and compound 10 were much less active. 
This suggests that replacement of the naphthalene core 
is also detrimental. Moreover compounds 9-11 are also 
inactive in the CK2 cellular assay showing that Naphth- 
2-ol is, so far, the sole substituent conferring activity in a 
cellular context. 

Collectively, it appeared from this SAR analysis 
that compound 4 was the most potent inhibitor in this 
compound series, being 10-fold more powerful than 
compound 1. Therefore, further studies were focused on 
compound 4. 

Analysis of CK2 inhibition by compound 4 

Dose-response curves for CK2 inhibition showed 
that compound 4 displayed an IC 50 ~0.4 uM on CK2a 
(Figure S2). To define the mode of CK2 inhibition by 



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compound 4, we performed steady-state kinetic analysis. 
It appeared that inhibition of CK2a by compound 4 was 
not competitive toward both ATP and peptide substrate 
(Figure 4). Again, plots were non linear with regards to 
peptide substrate concentrations. CK2 inhibition was 
strongly increased at high peptide substrate concentrations 
(Figure S3), a feature in line with the mode of inhibition 
of compound 1 (Figure ID and S3). 

Selectivity profiling of compound 4 

Specificity is a major concern for kinase inhibitor 
development. The kinase panel tested included members 
of major human protein kinase families [31] (Fig S4). This 
screening revealed that at 5uM, compound 4 inhibited 
CK2 by more than 95%, but had almost no effect on 
the other protein kinases tested (Table 2). Such high 
selectivity rules out compound 4 as promiscuous inhibitor. 
We calculated a previously described metric for kinase 
inhibitor selectivity based on the Gini coefficient [32]. 
The Gini score reflects, on a scale of 0 to 1 , the degree to 
which the inhibitory activity of a compound (calculated 
as the sum of inhibition of all kinases) is directed toward 
only a single kinase (a Gini score of 1) or is distributed 
equally across all tested kinases (a Gini score of 0). Not 
surprisingly, the calculated Gini coefficient for compound 
4 was 0.803 (Fig S5), a value among the highest for CK2 
inhibitors [33, 34] and for kinase inhibitors in general [35]. 
Such conclusion is also supported by the "hit rates" i.e. 
the number of kinases inhibited by >50% by compound 4 



using the 42 kinase panel: its value is 1, highlighting high 
specificity and suggesting that this compound might have 
characteristics of a uni-specific kinase inhibitor [35]. 

Analysis of compound 4 binding site 

Despite several attempts we could not get diffracting 
crystals to solve the 3D structure of a CK2-4 complex. 
Therefore, to delineate a potential binding site of compound 
4, we tested its inhibition on a series of CK2a mutants 
(Figure 5). Strikingly, this revealed that CK2 mutants that 
were found insensitive to compound 1, were also resistant 
to compound 4 inhibition. This strongly predicts that both 
compounds have a common binding site located on helix 
aC and on the activation segment of CK2a. 

Taken together this data indicates that compound 4 
and compound 1 have a common, or at least overlapping 
binding site and inhibit CK2 in a similar manner. 

Compound 1 and 4 promotes cell cycle arrest 

We then focus our attention on the U373 cell line 
because they were particularly responsive to compound 
1. These cells originate from glioblastoma, an aggressive 
tumor of the brain with poor survival prognostic. They 
harbor a p53 mutant isoform and are particularly resistant 
to drug-induced apoptosis [36]. First, we explored 
whether compound 1 could inhibit their cell cycle as it was 
already observed for compound 4 in endothelial cells [37] 
and A431 epidermoid carcinoma cells [38]. Cells were 




1/[ATP] (nM !) l/[Peptide] (nM 1 ) 



Figure 4: Characterization of compound 4 as a reversible non-competitive CK2 inhibitor. Lineweaver-Burk inhibition 
plots of human recombinant CK2a by compound 4. CK2 kinase activity was determined as described in the experimental section in the 
absence (♦) or in the presence of 0.2 (■), 0.4 ( ▲ ) and 0.8 (X) \iM compound 4 with various concentration of ATP (A) or peptide substrate 
(B). The data represent means of experiments run in triplicate with SEM never exceeding 10%. 



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treated with 50 (0.M of compound 1 for 48h or equivalent 
concentration of TBB, compounds 23, 4 or DMSO and 
then labeled with propidium iodide. U373 cells exposed 
to compounds 1 or 4 exhibited a cell cycle arrest with a 
substantial accumulation in mid-S-phase (Figure 6A). 

Azonaphthalene derivatives inhibit colony 
formation and tumor growth 

To assert the potential clinical interest of 
azonaphtalene derivatives, we first tested their ability to 
inhibit colony formation using soft agar assay as an in 
vitro surrogate of tumorigenesis. U373 cells can grow 
without anchorage and form colonies in soft agar culture 
reflecting their malignant properties. Colony formation 
assays were performed using compounds 1, 4, and 
compound 23 and TBB as controls. Both Compound 1 and 
4 inhibited colony formation in a dose dependent manner 
while TBB or compound 23 were without effect at the 
same concentration (Figure 6B). 

The NCI DTP website shows that compounds 1 and 
4 that were the most active in a colony formation assay, 
have a good pharmacological profile ( http://dtp.nci.nih. 
gov/docs/invivo/invivoscreen.html ) a property which was 



confirmed by the group of Atassi [38]. Compounds 3, 5 
and 6 were not evaluated by the NCI. 

We next tested the azonaphthalene compounds 
in a murine U373 tumor regression assay. We chose 
compound 4 for further in vivo investigations because 
of its high potency and specificity. The data illustrated in 
Figure 6C show that intratumoral injection of compound 
4 could decrease tumor incidence across all time points 
compared to mice injected with PBS. After 3 weeks, mice 
injected with compound 4 had tumors that were six times 
smaller than those injected with PBS (Figure 6C). Western 
blot analysis showed that Cdc37 Serl3 phosphorylation in 
U373 tumors, was significantly reduced in mice injected 
with compound 4 compared to mice injected with PBS 
(Figure 6C). 

To assess whether the anti-tumoral effect was 
related to CK2 inhibition, we wanted to inject mice 
with compound 23 that was used as inactive control in 
colony formation assay and CK2 activity cellular assay. 
However, a previous study [30] has shown that this 
compound displays antitumoral effect via inhibition 
of neoangiogenesis. Therefore, mice were treated with 
compound 9 (Calmagite) which was also inactive in the 
CK2 activity cellular assay. No significant anti-tumoral 



Table 2: Kinase Selectivity profile of compound 4. Residual kinase activity determined in the presence of 5 uM inhibitor is 
expressed as percentage of the control activity without inhibitor. Final concentration of ATP in the experiment was 100 |iM. 



Protein kinase 


Residual 
activity {%) 


Protein kinase 


Residual 
activity (%) 


Protein kinase 


Residual 
activity (%) 


Protein kinase 


Residual 
activity (%) 


CK2a2(h) 


4 


PKBa(h) 


95 


PDGFRa(h) 


106 


LOK(h) 


114 


PKA(h) 


57 


MKK7B(h) 


96 


ROCK-I(h) 


106 


CDK6/cyclinD3(h) 


115 


Lyn(h) 


63 


TAKl(h) 


96 


CDKl/cyclinB(h) 


109 


CHKl(h) 


121 


AMPKal(h) 


65 


c-RAF(h) 


97 


MEKl(h) 


110 


p70S6K(h) 


122 


CKlyl(h) 


82 


Pim-l(h) 


97 


PAK2(h) 


110 


ASKl(h) 


124 


MSTl(h) 


88 


eEF-2K(h) 


98 


CaMKI(h) 


111 


PKC6(h) 


128 


NEKll(h) 


89 


EGFR(h) 


98 


mTOR(h) 


111 


CDK7/cyclinH/MATl(h) 


129 


Plk3(h) 


90 


PKCa(h) 


100 


MKK6(h) 


112 


IRAK4(h) 


145 


DRAKl(h) 


94 


CDK2/cyclinA(h) 


101 


MLKl(h) 


112 


ALK(h) 


148 


JAK2(h) 


94 


EphA5(h) 


101 


KDR(h) 


113 






Fyn(h) 


95 


Abl(h) 


103 


IKKct(h) 


114 







1.5 



O 
+J 

TO 
i- 

>■ 

> 

u 



■a 

« 0.5 



0£ 



♦ 



51 



101 151 201 

Mutated residue 



251 



301 



Figure 5: Compound 4 shares overlapping binding 
site with compound 1. Inhibitory effect of compound 4 
on WT and a panel of mutants with the following residues 
mutated to alanine (10, 24, 61, 71, 74, 76, 80, 123, 138, 
160, 178, 195, 253, 268, 308, 311). Residual activity ratio 
is defined as the normalized activity of mutant CK2a 
divided by the normalized activity of wild-type CK2a 
in presence of 0.4|xM compound 4. Normalized activity 
represents the percentage of residual activity in presence 
of 0.4 |iM compound 4. 



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effect could be observed between mice treated with 
compound 9 and control vehicle (Figure S6). Collectively, 
these observations suggest that tumor growth inhibition 
by compound 4 is likely related to CK2 inhibition. 

DISCUSSION 

Abnormally high CK2 expression provides 
cancer cells with an environment favorable for tumor 
development [39]. Therefore development of CK2 
inhibitors may represent an interesting approach for 
the treatment of cancer. Most CK2 inhibitors are ATP- 
competitive but new development in the kinase field has 
raised interest in exploiting alternative mode of inhibition 
[40]. In this work, we describe a family of chemicals that 
are non-ATP competitive and cell potent CK2 inhibitors 
with good profile for their in vivo use. 



The exquisite selectivity of compound 4 revealed by 
its high Gini coefficient, could reflect unique features of 
CK2 and be related to the allosteric mechanism of action of 
this inhibitor. Although strong kinase selectivity may not 
be essential for efficacy of therapeutic agents, it is critical 
for tool compounds used to elucidate kinase biology. 

Site-specific mutations of Lys74Ala, Arg80Ala 
or Leul78A in CK2a showed that these mutant forms 
exhibit some resistance toward compound 1 and 4 
inhibition. Interestingly, these three mutations are 
located in or close to structural elements that are crucial 
for CK2 activity, namely the substrate binding site, the 
RD and DWG motifs [41, 42]. By interfering with these 
regulatory elements, compound 1 and 4 may stabilize a 
non-productive catalytic form of CK2a. 

We consistently observed parabolic inhibition plots in 
the presence of increasing peptide substrate concentrations, 
a feature that may arise from allosteric effects. In this 







Proportion of cells (%) in 






Sub-Gl 


Gl 


S 


G2/M 


DMSO 


1.1+0.09 


65.9±0.7 


7.5±1.1 


25.2±L9 


TBB 


2.1±0.02 


70.6±3.8 


3.2+0.3 


22.3±2.9 


1 


4.7±1.3 


61.2±0.7 


16±1.1 


17.4±1.4 


4 


4.3±0.9 


56.5±0.9 


19.6±1.2 


14.5±2.3 


23 


0.9±0.13 


75.3±0.5 


4.3±1 


22.3+2.1 





100 | 


0) 




'c 


80 


o 




o 




u 


60 


o 








01 
J2 


40 


E 








z 


20 


> 






0 


01 




cc 





0.5% 10|iM 25|iM 50|iM 50|iM 50|iM 50|aM 
DMSO 1 



TBB 



23 




III III 

4 or PBS 
Injections 



55 6b 
Days 

post -first injection 



Figure 6: Azonaphthalene derivatives promote cell cycle arrest, reduce colony formation and exert anti-tumoral effect 
in vivo. Nearly confluent U373 cells were treated with compounds or equivalent amount of DMSO. After 48h, cells were harvested and 
labeled with propidium iodide. DNA content was analysed with FACScalibur and Cell Quest software. Experiment was repeated three 
times. Proportion of U373 cells in sub-Gl, Gl, S and G2/M phases are summarized. B. Colony formation assay by soft agar culture. U373 
cells were poured on an agarose layer (0.6%) mixed with agarose (0.3%) containing compounds for colony formation assay (soft agar). 
Fifteen days later, colonies (more than 20 cells) were counted in 10 fields/well. Experiment was performed in duplicate at least three times. 
C. Athymic nude mice were inoculated subcutaneously into the right flank with 7.5xl0 5 U373 cells. When tumor reaches ± 50 mm 3 , animals 
were treated intratumorously 3 times weekly for 2 weeks, with compound 4 dissolved in PBS ( 1 mg/20 |il/injection) or PBS (control group). 
Tumor volume was determined twice weekly. Results represent the average of the tumor volume of 5 mice per group. Insert: representative 
western blots. Tumor extracts were analyzed by western blot for expression of tubulin, Cdc37 and P-Serl3-cdc37 [29]. 



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scenario, a substrate-dependent "conformation selection" 
would favor inhibitor binding. 

Our SAXS analysis showed that the overall shape 
of a CK2a-compound 1 complex is different from active 
CK2a shape obtained either by SAXS (this work) or 
by X-ray crystallography [43]. Moreover, the shape of 
the CK2a-compound 1 complex is also different from a 
structure of an inactive CK2a conformation [22]. Taken 
together, the binding of compound 1 may distort critical 
regulatory elements and lock the kinase in a new non- 
productive conformation. Further structural analysis will 
be required to precisely reveal the binding site of these 
inhibitors. 

Azonaphthalene derivatives have been described 
as nonpeptidyl thrombopoietin mimics [44]. We show 
that molecules belonging to this family of chemicals are 
cell-permeable and can target the cellular CK2 activity. 
A panel of tumor cell lines exposed to compound 1 
showed a decreased viability that was correlated with a 
down regulation of CK2 activity. Indeed, involvement of 
CK2 in cell cycle regulation is well documented. CK2 is 
required at multiple transitions in the cell cycle (including 
G0/G1, Gl/S and G2/M). We found that U373 cells 
exposed to compounds 1 or 4 exhibited a cell cycle arrest 
with a substantial accumulation in mid-S-phase. These 
observations are in accordance with similar effects of 
compound 4 previously described in endothelial cells [37]. 
Whether activation of apoptosis that occurs in HeLa cells 
treated with high doses of compound 1, is a consequence 
of a cell cycle arrest or direct promotion of apoptosis 
remains to be established. Of note, the viability of U373 
cells which are particularly resistant to drug-induced 
apoptosis [36] was strongly affected by compound 1 and 
this effect was correlated with a down regulation of CK2 
activity. 

Compound 1 inhibited U373 cell colony formation in 
a dose dependent manner. Of note, CK2 phosphorylation of 
HSP90 modulates chaperone function and drug sensitivity 
[45]. By promoting activities of several oncokinases, 
the Cdc37/Hsp90 chaperone complex contributes to the 
acceleration of cell proliferation observed in cancer cells. 
Like CK2, Cdc37 is over-expressed in cancer cells and a 
target for cancer therapy [46]. To interact properly with 
client kinases, Cdc37 must be phosphorylated on Serl3 
by CK2. We showed that compound 1 decreases Cdc37 
phosphorylation on Serl3 both in U373 cells and in 
tumor-bearing mice. We can hypothesize that at least part 
of the anti tumorigenic effect of this class of compound 
could involve inhibition of Cdc37 phosphorylation. 
Interestingly, it was recently reported that Sarcoma 180 
tumors were sensitive to compound 1 showing a -50% 
tumor regression in treated mice (NCI DTP website). 
Cdc37 Serl3 phosphorylation in glioblastoma tumors was 
also significantly reduced in mice injected with compound 
4 suggesting that at least part of the antitumoral effect of 
this class of inhibitors could involve inhibition of Cdc37. 



We cannot exclude that the effect of azonaphthalene 
derivatives could be also explained by the partial inhibition 
of other targets. However, to date compound 4 is among 
the most selective CK2 inhibitors published. 

In this work, we showed that compound 4 induced a 
significant inhibition of glioblastoma tumor growth that was 
correlated with CK2 inhibition. Of note, compound 4 was 
reported to inhibit angiogenesis in a chick chorioallantoic 
membrane assay and proliferation of primary endothelial 
cells, A431, L1210 and M5076 cancer cells [38]. In this 
study, the authors suggested that the effect of compound 
4 could involve cell cycle perturbation through inhibition 
of topoisomerase II catalytic activity [38]. Interestingly, 
topoisomerase II is a known CK2 target [47]. 

In conclusion, we described the discovery of a 
family of Azonaphthalene derivatives that perturb CK2a 
conformation thereby blocking productive binding 
of substrates. These compounds may be useful as 
research tools in probing CK2 conformational plasticity 
because our kinetic analysis showed a mechanism of 
action consistent with an allosteric mode of inhibition, 
highlighting the opportunity of exploiting different CK2 
inhibition mechanisms [19]. Although further structural 
analysis will be required to reveal the binding site of these 
inhibitors, their therapeutic potential is emphasized by 
their efficacy in growth inhibition of apoptosis-resistant 
tumors. Further optimization of these compounds and the 
results from further in vivo testing may aid in the design 
of other classes of CK2 inhibitors and should reveal their 
efficacy in additional specific models. 

METHODS 



Cell culture 

HeLa (cervical adenocarcinoma), U373 
(glioblastoma) and MDA23 1 (breast adenocarcinoma) cell 
lines were cultivated in Dulbecco's medium (Invitrogen) 
Life Technologies, Inc.) while A549 (lung carcinoma), 
HI 299 (lung carcinoma) and LnCap (prostate carcinoma) 
cell lines were cultivated in RPMI (Invitrogen) Life 
Technologies, Inc.). Each medium was supplemented with 
10% (v/v) fetal calf serum (FBS, Bio West). MCF7 (breast 
adenocarcinoma) cell line was cultivated in Dulbecco's 
medium with insulin (10|ig/ml). 

High-throughput screening 

High-throughput screening of the NCI Diversity 
Set chemical library (2,860 compounds) was performed 
as previously described [48]. Active fractions from the 
primary screen were retested from freshly made solutions. 



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Cellular CK2 activity assay 



CK2a production and purification 



A cellular CK2 activity assay was performed as 
previously described [28]. 

Western Blot 

Western-blotting was performed as previously 
described [11] using the following antibodies: anti-CK2a 
antibody [3] at 1/5000, oranti-Serl3-phospho Cdc37 [29] 
at 1/3000, anti-Cdc37 (Santa Cruz, scl3129) at 1/2000 
and anti-tubulin (YL1/2, Abeam) at 1/50000. 

Kinase selectivity profiling 

Kinase selectivity of compound 4 was assessed using 
the Kinase Profiler service offered by Millipore which 
utilizes a radiometric filter-binding assay. The assays 
were performed at 100 uM ATP in the presence of 5 uM 
inhibitor. Inhibition, expressed as the percent of activity 
determined in the absence of inhibitor, was calculated 
from the residual activity measured in the presence of 5 
uM inhibitor. 

Selectivity parameters 

Lorenz curves were derived from the selectivity 
data. Gini coefficients and Hit rates were calculated as 
described in [32]. 

Cell Viability 

Cell viability assay was performed as described 
previously [28] 

Cell cycle distribution analysis 

Cell cycle distribution analysis was performed as 
described previously [15]. 

Soft agar assay 

Soft agar assay was performed as described 
previously [15]. 

CK2 Phosphorylation assays 

CK2 radiometric kinase assay based on conventional 
filter-binding assay was performed as previously described 
[20]. 



GST-rhCK2a AC (l-335) expression in E.Coli BL21 
cells after induction with IPTG and lysis were performed 
as described in [15]. Supernatant was added onto 
glutathion-sepharose beads (Amersham). After overnight 
incubation, beads were washed by 40mM Tris pH 7.5, 
150 mM NaCl and cleavage reaction was performed 
on column (1 unit of thrombin.mL A of beads (Sigma, 
T6634) in PBS containing 200 mM NaCl, 2% glycerol 
and 1 mM DTT for 4h at 4°C). Following elution (50 mM 
Tris pH 7.5, 150 mM NaCl) the proteins were loaded on 
a heparine-sepharose column equilibrated in the elution 
buffer. Proteins were then eluted with a linear gradient of 
0. 15-1M NaCl in 50 mM Tris pH 7.5. The active fractions 
were pooled and concentrated with a Centricon Plus-70mL 
(Millipore). Production and purification of GST-rhCK2a 
mutants were carried out using the same protocol. 

SAXS experiments 

SAXS experiments were performed at the SOLEIL 
Synchrotron (Saclay, France) on beamline SWING. The 
wavelength X was 1.0 A (1 A=0.1 nm) and the sample-to- 
detector distance was 1924 mm giving access to scattering 
vectors q ranging from 0.009 to 0.47 A-l.The scattering 
vector is defined as q = 4n IX sin8, where 28 is the 
scattering angle. The detector was an AVIEX170170 CCD 
detector, and 40 successive frames of 4.2 ms exposure 
were recorded for each sample. The samples were injected 
with an automatic sample-changer and circulated through 
an evacuated quartz capillary between each frame to avoid 
radiation damage. 

Protein concentration of CK2a (in 25 mM Tris pH 
8.5, 0.2 M NaCl, 1 mM DTT and 10% glycerol) was 
varied from 1.25 to 10 nig-mF 1 in order to check for 
interparticle interactions. The protein concentration of 
CK2a in the presence of 500 uM compound 1 (CK2a/l 
ratio 1:2) was varied from 1.25 to 10 mg mr 1 . The radius 
of gyration R Q was derived by the Guinier approximation 
I(q)=I(0yexp(-q 2 R G 2 /3) for qR a <l.0. The distance 
distribution function P(f) was calculated by the Fourier 
inversion of the scattering intensity I(q) using GNOM 
[491. The maximum diameter of the macromolecule, D , 

L J 3 max 3 

was also determined [50]. 

Using the program GASBOR [51], the overall shapes 
of CK2a alone or in complex with compound 1 were 
restored from the experimental data. Ten independent fits 
were run with no symmetry restriction, and the stability of 
the solution was checked. The atomic structure of CK2a 
(PDB ID 1PJK) was then fitted in the averaged calculated 
shape, using SUPCOMB [52]. The theoretical scattering 
curve of CK2a (PDB ID 1PJK) was calculated using 
CRYSOL [53]. 



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Murine xenograft tumor growth assays 

All experimental procedures adhered to our local 
ethical committee (Comite regional d'efhique pour 
l'Experimentation animale CREEA, Rhone Alpes - 
protocol n°315). Female Harlan athymic nude mice (6-8 
weeks) were inoculated subcutaneously into the right 
flank with 7.5xl0 5 U373 cells. When tumors reaches ± 50 
mm 3 (volume = length x width x height), animals were 
treated intratumorously 3 times weekly for 2 weeks, with 
compound 4 dissolved in PBS (1 mg/20 Lil/injection) or 
PBS (control group). Body weight and tumor volume 
were determined twice weekly. The experiment was 
terminated when tumor volume was about 1000 mm 3 . 
Data are expressed as means and s.d. and were analysed 
with Student's ?-test; significance is defined as /?<0.05. 

Tumor extracts were prepared in RIPA buffer and 
analyzed by western blotting. 

ABBREVIATIONS 

CK2: Casein Kinase 2; SAXS: Small Angle X-ray 
Scattering ; TBB : 4,5,6,7-TetraBromo-lH-Benzotriazole 
; R Q : radius of gyration. 

ACKNOWLEDGMENTS 

The authors thank Dr. V. Colin for her help during 
FACS experiment and H. Pointu and his staff for animal 
health care. Thanks also to the ChemAxon company ( http:// 
www.chemaxon.com ) which has allowed the academic 
Tamis software team to freely use the MarvinView 
package, We acknowledge SOLEIL for access to the 
Beamline Swing and Javier Perez for assistance. The 
authors gratefully thank the National Cancer Institute 
Developmental Therapeutics Program for providing 
chemical libraries. 

GRANT SUPPORT 

This work was supported by the Institut National 
de la Sante et de la Recherche Medicale (INSERM), the 
Centre National pour la Recherche Scientifique (CNRS), 
the Commissariat a l'Energie atomique (CEA), the Institut 
Curie, the Ligue Nationale Contre le Cancer (equipe 
labellisee 2010), the Institut National du Cancer (grant 
number 57). RP gratefully acknowledges support from the 
Fondation pour la Recherche Medicale Fellowship. 

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